Conjugated double strand compositions for use in gene modulation   

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority benefit to U.S. Provisional Application No. 60/786,570, filed Mar. 27, 2006 and entitled, “Double Strand Compositions comprising differentially Modified Strands for use in Gene Modulation” the entirety of the disclosure is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention provides compositions comprising oligomeric compounds that modulate gene expression. In one embodiment, such modulation is via the RNA interference pathway. The modified oligomeric compounds of the invention comprise motifs that can enhance various physical properties and attributes compared to wild type nucleic acids. More particularly, the modification of both strands enables enhancing each strand independently for maximum efficiency for their particular roles in a selected pathway such as the RNAi pathway. The compositions of the present invention further include a linked conjugate group and varying numbers of phosphorothioate internucleoside linkages that enhance the in vivo activity. The compositions are useful for, for example, targeting selected nucleic acid molecules and modulating the expression of one or more genes. In some embodiments, the compositions of the present invention hybridize to a portion of a target RNA resulting in loss of normal function of the target RNA.

BACKGROUND OF THE INVENTION

In many species, introduction of double-stranded RNA (dsRNA) induces potent and specific gene silencing. This phenomenon occurs in both plants and animals and has roles in viral defense and transposon silencing mechanisms. This phenomenon was originally described more than a decade ago by researchers working with the petunia flower. While trying to deepen the purple color of these flowers, Jorgensen et al. introduced a pigment-producing gene under the control of a powerful promoter. Instead of the expected deep purple color, many of the flowers appeared variegated or even white. Jorgensen named the observed phenomenon “cosuppression”, since the expression of both the introduced gene and the homologous endogenous gene was suppressed (Napoli et al., Plant Cell, 1990, 2, 279-289; Jorgensen et al., Plant Mol. Biol., 1996, 31, 957-973).

Cosuppression has since been found to occur in many species of plants, fungi, and has been particularly well characterized in Neurospora crassa, where it is known as “quelling” (Cogoni et al., Genes Dev., 2000, 10, 638-643; Guru, Nature, 2000, 404, 804-808).

The first evidence that dsRNA could lead to gene silencing in animals came from work in the nematode, C. elegans. In 1995, researchers Guo and Kemphues were attempting to use antisense RNA to shut down expression of the par-1 gene in order to assess its function. As expected, injection of the antisense RNA disrupted expression of par-1, but quizzically, injection of the sense-strand control also disrupted expression (Guo et al., Cell, 1995, 81, 611-620). This result was a puzzle until Fire et al. injected dsRNA (a mixture of both sense and antisense strands) into C. elegans. This injection resulted in much more efficient silencing than injection of either the sense or the antisense strands alone. Injection of just a few molecules of dsRNA per cell was sufficient to completely silence the homologous gene\'s expression. Furthermore, injection of dsRNA into the gut of the worm caused gene silencing not only throughout the worm, but also in first generation offspring (Fire et al., Nature, 1998, 391, 806-811).

The potency of this phenomenon led Timmons and Fire to explore the limits of the dsRNA effects by feeding nematodes bacteria that had been engineered to express dsRNA homologous to the C. elegans unc-22 gene. Surprisingly, these worms developed an unc-22 null-like phenotype (Timmons et al., Nature, 1998, 395, 854; Timmons et al., Gene, 2001, 263, 103-112). Further work showed that soaking worms in dsRNA was also able to induce silencing (Tabara et al., Science, 1998, 282, 430-431). PCT publication WO 01/48183 discloses methods of inhibiting expression of a target gene in a nematode worm involving feeding to the worm a food organism which is capable of producing a double-stranded RNA structure having a nucleotide sequence substantially identical to a portion of the target gene following ingestion of the food organism by the nematode, or by introducing a DNA capable of producing the double-stranded RNA structure.

The posttranscriptional gene silencing defined in C. elegans resulting from exposure to double-stranded RNA (dsRNA) has since been designated as RNA interference (RNAi). This term has come to generalize all forms of gene silencing involving dsRNA leading to the sequence-specific reduction of endogenous targeted mRNA levels; unlike co-suppression, in which transgenic DNA leads to silencing of both the transgene and the endogenous gene.

Introduction of exogenous double-stranded RNA (dsRNA) into C. elegans has been shown to specifically and potently disrupt the activity of genes containing homologous sequences. Montgomery et al. suggests that the primary interference effects of dsRNA are post-transcriptional; this conclusion being derived from examination of the primary DNA sequence after dsRNA-mediated interference a finding of no evidence of alterations followed by studies involving alteration of an upstream operon having no effect on the activity of its downstream gene. These results argue against an effect on initiation or elongation of transcription. Finally they observed by in situ hybridizations that dsRNA-mediated interference produced a substantial, although not complete, reduction in accumulation of nascent transcripts in the nucleus, while cytoplasmic accumulation of transcripts was virtually eliminated. These results indicate that the endogenous mRNA is the primary target for interference and suggest a mechanism that degrades the targeted mRNA before translation can occur. It was also found that this mechanism is not dependent on the SMG system, an mRNA surveillance system in C. elegans responsible for targeting and destroying aberrant messages. The authors further suggest a model of how dsRNA might function as a catalytic mechanism to target homologous mRNAs for degradation. (Montgomery et al., Proc. Natl. Acad. Sci. U S A, 1998, 95, 15502-15507).

The development of a cell-free system from syncytial blastoderm Drosophila embryos that recapitulates many of the features of RNAi has been reported. The interference observed in this reaction is sequence specific, is promoted by dsRNA but not single-stranded RNA, functions by specific mRNA degradation, and requires a minimum length of dsRNA. Furthermore, preincubation of dsRNA potentiates its activity demonstrating that RNAi can be mediated by sequence-specific processes in soluble reactions (Tuschl et al., Genes Dev., 1999, 13, 3191-3197).

In subsequent experiments, Tuschl et al., using the Drosophila in vitro system, demonstrated that 21- and 22-nt RNA fragments are the sequence-specific mediators of RNAi. These fragments, which they termed short interfering RNAs (siRNAs) were shown to be generated by an RNase III-like processing reaction from long dsRNA. They also showed that chemically synthesized siRNA duplexes with overhanging 3′ ends mediate efficient target RNA cleavage in the Drosophila lysate, and that the cleavage site is located near the center of the region spanned by the guiding siRNA. In addition, they suggest that the direction of dsRNA processing determines whether sense or antisense target RNA can be cleaved by the siRNA-protein complex (Elbashir et al., Genes Dev., 2001, 15, 188-200). Further characterization of the suppression of expression of endogenous and heterologous genes caused by the 21-23 nucleotide siRNAs have been investigated in several mammalian cell lines, including human embryonic kidney (293) and HeLa cells (Elbashir et al., Nature, 2001, 411, 494-498).

Tijsterman et al. have shown that, in fact, single-stranded RNA oligomers of antisense polarity can be potent inducers of gene silencing. As is the case for co-suppression, they showed that antisense RNAs act independently of the RNAi genes rde-1 and rde-4 but require the mutator/RNAi gene mut-7 and a putative DEAD box RNA helicase, mut-4. According to the authors, their data favor the hypothesis that gene silencing is accomplished by RNA primer extension using the mRNA as template, leading to dsRNA that is subsequently degraded suggesting that single-stranded RNA oligomers are ultimately responsible for the RNAi phenomenon (Tijsterman et al., Science, 2002, 295, 694-697).

Several other publications have described the structural requirements for the dsRNA trigger required for RNAi activity. Recent reports have indicated that ideal dsRNA sequences are 21 nt in length containing 2 nt 3′-end overhangs (Elbashir et al, EMBO (2001), 20, 6877-6887, Sabine Brantl, Biochimica et Biophysica Acta, 2002, 1575, 15-25.) In this system, substitution of the 4 nucleosides from the 3′-end with 2′-deoxynucleosides has been demonstrated to not affect activity. On the other hand, substitution with 2′-deoxynucleosides or 2′-OMe-nucleosides throughout the sequence (sense or antisense) was shown to be deleterious to RNAi activity.

Investigation of the structural requirements for RNA silencing in C. elegans has demonstrated modification of the internucleotide linkage (phosphorothioate) to not interfere with activity (Parrish et al., Molecular Cell, 2000, 6, 1077-1087.) It was also shown by Parrish et al., that chemical modification like 2′-amino or 5′-iodouridine are well tolerated in the sense strand but not the antisense strand of the dsRNA suggesting differing roles for the 2 strands in RNAi. Base modification such as guanine to inosine (where one hydrogen bond is lost) has been demonstrated to decrease RNAi activity independently of the position of the modification (sense or antisense). Same “position independent” loss of activity has been observed following the introduction of mismatches in the dsRNA trigger. Some types of modifications, for example introduction of sterically demanding bases such as 5-iodoU, have been shown to be deleterious to RNAi activity when positioned in the antisense strand, whereas modifications positioned in the sense strand were shown to be less detrimental to RNAi activity. As was the case for the 21 nt dsRNA sequences, RNA-DNA heteroduplexes did not serve as triggers for RNAi. However, dsRNA containing 2′-F-2′-deoxynucleosides appeared to be efficient in triggering RNAi response independent of the position (sense or antisense) of the 2′-F-2′-deoxynucleosides.

In one experiment the reduction of gene expression was studied using electroporated dsRNA and a 25mer morpholino in post implantation mouse embryos (Mellitzer et al., Mehanisms of Development, 2002, 118, 57-63). The morpholino oligomer did show activity but was not as effective as the dsRNA.

A number of PCT applications have been published that relate to the RNAi phenomenon. These include: PCT publication WO 00/44895; PCT publication WO 00/49035; PCT publication WO 00/63364; PCT publication WO 01/36641; PCT publication WO 01/36646; PCT publication WO 99/32619; PCT publication WO 00/44914; PCT publication WO 01/29058; and PCT publication WO 01/75164.

U.S. Pat. Nos. 5,898,031 and 6,107,094 describe certain oligonucleotide having RNA like properties. When hybridized with RNA, these oligonucleotides serve as substrates for a dsRNase enzyme with resultant cleavage of the RNA by the enzyme.

In another published paper (Martinez et al., Cell, 2002, 110, 563-574) it was shown that double stranded as well as single stranded siRNA resides in the RNA-induced silencing complex (RISC) together with elF2C1 and elf2C2 (human GERp950 Argonaute proteins. The activity of 5′-phosphorylated single stranded siRNA was comparable to the double stranded siRNA in the system studied. In a related study, the inclusion of a 5′-phosphate moiety was shown to enhance activity of siRNA\'s in vivo in Drosophila embryos (Boutla, et al., Curr. Biol., 2001, 11, 1776-1780). In another study, it was reported that the 5′-phosphate was required for siRNA function in human HeLa cells (Schwarz et al., Molecular Cell, 2002, 10, 537-548).

A wide variety of chemical modifications have been made to siRNA compositions to try to enhance properties including stability and potency relative to the unmodified compositions. Much of the early work looked at modification of one strand while keeping the other strand unmodified. More recent work has focused on modification of both strands.

One group is working on modifying both strands of siRNA duplexes such that each strand has an alternating pattern wherein each nucleoside or a block of modified nucleosides is alternating with unmodified β-D-ribonucleosides. The chemical modification used in the modified portion is 2′-OCH3 modified nucleosides (see European publication EP 1389637 A1, published on Feb. 18, 2004 and PCT publication WO2004015107 published on Feb. 19, 2004).

Another group has prepared a number of siRNA constructs with modifications in both strands (see PCT publication WO03/070918 published on Aug. 28, 2003). The constructs disclosed generally have modified nucleosides dispersed in a pattern that is dictated by which strand is being modified and further by the positioning of the purines and pyrimidines in that strand. In general the purines are 2′-OCH3 or 2′-H and pyrimidines are 2′-F in the antisense strand and the purines are 2′-H and the pyrimidines are 2′-OCH3 or 2′-F in the sense strand. According to the definitions used in the present application these constructs would appear to be positionally modified as there is no set motif to the substitution pattern and positionally modified can describe a random substitution pattern.

Certain nucleoside compounds having bicyclic sugar moieties are known as locked nucleic acids or LNA (Koshkin et al., Tetrahedron 1998, 54, 3607-3630). These compounds are also referred to in the literature as bicyclic nucleotide analogs (Imanishi et al., International Patent Application WO 98/39352), but this term is also applicable to a genus of compounds that includes other analogs in addition to LNAs. Such modified nucleosides mimic the 3′-endo sugar conformation of native ribonucleosides with the advantage of having enhanced binding affinity and increased resistance to nucleases.

One group recently reported that the incorporation of bicyclic nucleosides, each having a 4′-CH2—O-2′ bridge (LNA) into siRNA duplexes dramatically improved the half life in serum via enhanced nuclease resistance and also increased the duplex stability due to the increased affinity. This effect is seen with a minimum number of LNA\'s located as specific positions within the siRNA duplex. The placement of LNA\'s at the 5′-end of the sense strand was shown to reduce the loading of this strand which reduces off target effects (see Elmen et al., Nucleic Acids Res., 2005, 33(1), 439-447).

Some LNAs have a 2′-hydroxyl group linked to the 4′ carbon atom of the sugar ring thereby forming a bicyclic sugar moiety. The linkage may be a methylene (—CH2—)n group bridging the 2′ oxygen atom and the 4′ carbon atom wherein n is 1 or 2 (Singh et al., Chem. Commun., 1998, 4, 455-456; Kaneko et al., U.S. Patent Application Publication No. US 2002/0147332, also see Japanese Patent Application HEI-11-33863, Feb. 12, 1999).

U.S. Patent Application Publication No. 2002/0068708 discloses a number of nucleosides having a variety of bicyclic sugar moieties with the various bridges creating the bicyclic sugar having a variety of configurations and chemical composition.

Braash et al., Biochemistry 2003, 42, 7967-7975 report improved thermal stability of LNA modified siRNA without compromising the efficiency of the siRNA. Grunweller, et. al., Nucleic Acid Research, 2003, 31, 3185-3193 discloses the potency of certain LNA gapmers and siRNAs.

One group has identified a 9 base sequence within an siRNA duplex that elicits a sequence-specific TLR7-dependent immune response in plasmacytoid dendritic cells. The immunostimulation was reduced by incorporating 4 bicyclic nucleosides, each having a 4′-CH2—O-2′ bridge (LNA) at the 3′-end of the sense strand. They also made 5′ and both 3′ and 5′ versions of sense and antisense for incorporation into siRNA duplexes where one strand had the modified nucleosides and the other strand was unmodified (see Hornung et al., 2005, 11(3)I, 263-270).

One group of researchers used expression profiling to perform a genome wide analysis of the efficacy and specificity of siRNA induced silencing of two genes involved in signal transduction (insulin-like growth factor receptor (IGF1R) and mitogen-activated protein kinase 1 (MAPK14 or p38α). A unique expression profile was produced for each of the 8 siRNAs targeted to MAPK14 and 16 siRNA\'s targeted to IGF1R indicating that off target effects were highly dependent on the particular sequence. These expression patterns were reproducable for each individual siRNA. The group determined that off target effects were caused by both the antisense strand and the sense strand of siRNA duplexes. There is a need for siRNA\'s that are designed to preferentially load only the antisense strand thereby reducing the off target effects caused by the sense strand also being loaded into the RISC.

A number of published applications that are commonly assigned with the present application disclose double strand compositions wherein one or both of the strands comprise a particular motif. The motifs include hemimer motifs, blockmer motifs, gapped motifs, fully modified motifs, positionally modified motifs and alternating motifs (see published PCT applications: WO 2004/044133 published May 27, 2004, 3′-endo motifs; WO 2004/113496 published Dec. 29, 2004, 3′-endo motifs; WO 2004/044136 published May 27, 2004, alternating motifs; WO 2004/044140 published May 27, 2004, 2′-modified motifs; WO 2004/043977 published May 27, 2004, 2′-F motifs; WO 2004/043978 published May 27, 2004, 2′-OCH3 Motifs; WO 2004/041889 published May 21, 2004, polycyclic sugar motifs; WO 2004/043979 published May 27, 2004, sugar surrogate motifs; and WO 2004/044138 published May 27, 2004, chimeric motifs; also see published US Application US20050080246 published Apr. 14, 2005).

Like the RNAse H pathway, the RNA interference pathway of antisense modulation of gene expression is an effective means for modulating the levels of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications involving gene silencing. The present invention therefore further provides compositions useful for modulating gene expression pathways, including those relying on an antisense mechanism of action such as RNA interference and dsRNA enzymes as well as non-antisense mechanisms. One having skill in the art, once armed with this disclosure will be able, without undue experimentation, to identify additional compositions for these uses.

SUMMARY

In one embodiment, the present invention provides compositions comprising first and second chemically synthesized oligomeric compounds, wherein:

said first oligomeric compound comprises a hybridizing region that is essentially fully complementary to a nucleic acid target, a non hybridizing 3′-overhang region, a 5′-phosphate moiety and wherein from 2 to about 10 of the internucleoside linkages are phosphorothioate internucleoside linkages;

said second oligomeric compound comprises a hybridizing region that is essentially fully complementary to the hybridizing region of the first oligomeric compound, an optional non hybridizing 3′-overhang region and a conjugate group that is attached to a phosphorothioate group by a bivalent linking group where the phosphorothioate group is attached to the 3′-end of the oligomeric compound and wherein from 1 to about 10 of the internucleoside linkages are phosphorothioate internucleoside linkages;

each of said hybridizing regions independently comprises a contiguous sequence of from 17 to 21 nucleosides linked by internucleoside linking groups wherein each sequence independently defines a motif selected from an alternating motif, a gapped motif, a positional motif or a fully modified motif; and

each of said 3′-overhang regions independently comprises from 1 to 3 2′-modified nucleosides wherein at least one of the 2′-modified nucleosides have enhanced nuclease resistance relative to a β-D-2′-deoxyribonucleoside and wherein each internucleoside linking group between nucleosides in the overhang regions and between the overhang regions and the hybridizing regions are phosphorothioate internucleoside linking groups.

In one embodiment the hybridizing regions of the first and second oligomeric compounds are each 19 nucleosides in length. In another embodiment the hybridizing region of the second oligomeric compound is fully complementary to the first oligomeric compound. In a further embodiment the hybridizing region of the first oligomeric compound is fully complementary to a nucleic acid target.

In one embodiment the non hybridizing overhang region of the first oligomeric compound has 1 or 2 2′-modified nucleosides. In another embodiment the non hybridizing overhang region of the first oligomeric compound has 2 2′-modified nucleosides.

In one embodiment the second oligomeric compound does not have a non hybridizing overhang region.

In one embodiment the non hybridizing overhang region of the second oligomeric compound has 1 or 2 2′-modified nucleosides. In another embodiment the non hybridizing overhang region of the second oligomeric compound has 2 2′-modified nucleosides.

In one embodiment only one of the nucleosides in at least one of the overhang regions has enhanced nuclease resistance relative to a β-D-2′-deoxyribonucleoside.

In one embodiment each 2′-modified nucleoside, of each non hybridizing overhang region, comprises a 2′-substituent group independently selected from —O—C1-6-alkyl, substituted —O—C1-6-alkyl, substituted (—O—C1-6-alkyl)2, —O—C2-6-alkenyl, substituted —O—C2-6-alkenyl, —O—C2-6-alkynyl, substituted —O—C2-6-alkynyl, substituted —O-acetamide (—O—CH2C)═O)—N(—)2) or allyl; wherein each substituent group is, independently, halogen, —O—, —N(R1)— or —S—C1-6-alkyl; substituted —O—, —N(R1)—, or —S—C1-6-alkyl, —O—, —N(R)—, or —S—C2-6-alkenyl, substituted —O—, —N(R1)—, or —S—C2-6-alkenyl; —O—N(R1)(R2) or —N(R1)(R2); and each R1 and R2 is, independently, H, —C1-6-alkyl, substituted —C1-6-alkyl or an amino protecting group. In another embodiment each 2′-substituent group is, independently, —O—C1-4 alkyl, —OCF3, —O—(CH2)2—OCH3, —O—(CH2)2—SCH3, —O—(CH2)3—NH2, —CH7—C(H)═CH2—O—CH2—C(H)═CH2, —O—CH2)2—O—N(R1)(R2), —O—CH2—C(═O)—N(R1)(R2) or —O—(CH2)2—O—(CH2)2—N(R1)(R2) where each R1 and R2 is, independently, H, —C1-6-alkyl, substituted —C1-6-alkyl or an amino protecting group. In a further embodiment each 2′-substituent group is, independently, —O—C1-C3 alkyl, —O—(CH2)2—OCH3, —O—(CH2)2—O—N(CH3)2, —O—CH2—C(═O)—N(H)(CH3), —O—CH2—C(═O)—N(H)(CH2)2N(CH3)2 or —O—CH2)2—O—(CH2)2—N(CH3). In a more preferred embodiment each 2′-substituent group is, independently, —O—CH3, —O—(CH2)2—OCH3, —O—(CH2)2—O—N(CH3)2 or O—CH2—C(═O)—N(H)(CH3). In another preferred embodiment each 2′-substituent group is —O—(CH2)2—OCH3.

In one embodiment the first oligomeric compound has from about 6 to about 8 phosphorothioate internucleoside linkages. In another embodiment the first oligomeric compound has 7 phosphorothioate internucleoside linkages.

In one embodiment the phosphorothioate internucleoside linkages of the first oligomeric compound are essentially consecutively located starting from the 3′-end.

In one embodiment the second oligomeric compound comprises from 1 to about 10 phosphorothioate internucleoside linkages. In another embodiment the second oligomeric compound comprises from 1 to about 7 phosphorothioate internucleoside linkages. In a further embodiment the second oligomeric compound comprises about 7 phosphorothioate internucleoside linkages.

In one embodiment the sequence of the hybridizing region of the first oligomeric compound defines an alternating motif. In another embodiment the sequence of the hybridizing region of the first oligomeric compound defines a fully modified motif. In a further embodiment the sequence of the hybridizing region of the first oligomeric compound defines a positional motif. In another embodiment the sequence of the hybridizing region of the first oligomeric compound defines a gapped motif.

In one embodiment the sequence of the hybridizing region of the second oligomeric compound defines an alternating motif. In another embodiment the sequence of the hybridizing region of the second oligomeric compound defines a gapped motif. In a further embodiment the sequence of the hybridizing region of the second oligomeric compound defines a positional motif. In another embodiment the sequence of the hybridizing region of the second oligomeric compound defines a fully modified motif.

In one embodiment the conjugate is selected from peptides, proteins, sterols, lipids, phospholipids, biotin, phenoxazines, an active drug substance or folates. In another embodiment the conjugate is cholesterol or a lipid. In a further embodiment the conjugate is cholesterol. In another embodiment the lipid is a C8-C18 g lipid. In a further embodiment the lipid is fully unsaturated, fully saturated or partially saturated. In another embodiment the lipid is myristic acid, oleic acid omega 3 or C16. In a further embodiment the conjugate is an active drug substance. In another embodiment the conjugate is aspirin or ibuprofcn. In another embodiment the conjugate is octreotate or lyp-1 protein.

The present invention also provides methods of inhibiting gene expression comprising contacting one or more cells, a tissue or an animal with any composition described herein.

The present invention provides compositions having first and second oligomeric compounds wherein the first oligomeric compound comprises from 1 to 3 non hybridizing nuclease resistant 2′-modified nucleosides at the 3′-end and the second oligomeric compound comprises a conjugate group. Each of the oligomeric compounds have from 1 to about 10 phosphorothioate internucleoside linkages. The sequence of linked nucleosides in the hybridizing region of each oligomeric compound independently defines a specific motif. The motifs derive from the positioning of modified nucleosides relative to other modified or unmodified nucleosides in a strand and are independent of the sequence or type of nucleobases (purine, pyrimidine or other) or the internucleoside linkages. The compositions of the present invention include those that are differentially modified wherein each strand comprises a different motif and also include compositions wherein the motif is the same in each strand but compositions having identical motifs will normally have chemical variations or alignments that will differentiate the strands.

In one embodiment of the present invention compositions are provided comprising first and second oligomeric compounds wherein each oligomeric compound independently has a hybridizing region of from about 17 to about 21 nucleosides wherein essentially each of the nucleosides of the second oligomeric compound are complementary to and hybridize to the first oligomeric compound. In a preferred embodiment the second oliogmeric compound is fully complementary to the first oliogmeric compound. In another embodiment one or more mismatches, modified nucleosides (e.g. abasic, acyclic et al.,) or non nucleosides are also contemplated.

The first oligomeric compound further includes a non hybridizing 3′-overhang region comprising from 1 to 3 2′-modified nucleosides. At least one of these non hybridizing 2′-modified nucleosides has increased nuclease resistance relative to a β-D-2′-deoxyribonucleoside being located at the same position. In a more preferred embodiment all of the non hybridizing 2′-modified nucleosides have increased nuclease resistance relative to a β-D-2′-deoxyribonucleoside being located at the same position. Such 2′-modified nucleosides can be further modified such as for example 4′-S and/or 5′-modified. The 2′-modified nucleosides further include 2′-substituted nucleosides as well as bicyclic nucleosides (wherein one point of attachment for the second ring is the 2′-position such as for example 4′-(CH2)n—O-2′ where n is 1 or 2). In one embodiment the non hybridizing 3′-overhang region comprises 2′-modified nucleosides. In one embodiment the first oligomeric compound has a 5′-hydroxyl or 5′-protected hydroxyl group. In a preferred embodiment the first oligomeric compound includes a 5′-phosphate group.

The second oligomeric compound further includes an optional non hybridizing 3′-overhang region comprising from 1 to 3 2′-modified nucleosides. At least one of these non hybridizing 2′-modified nucleosides, when present, has increased nuclease resistance relative to a β-D-2′-deoxyribonucleoside being located at the same position. In a more preferred embodiment all of the non hybridizing 2′-modified nucleosides have increased nuclease resistance relative to a β-D-2′-deoxyribonucleoside being located at the same position. Such 2′-modified nucleosides can be further modified such as for example 4′-S and/or 5′-modified. The 2′-modified nucleosides further include 2′-substituted nucleosides as well as bicyclic nucleosides (wherein one point of attachment for the second ring is the 2′-position such as for example 4′-(CH2)n—O-2′ where n is 1 or 2). In one embodiment the non hybridizing 3′-overhang region comprises 2′-modified nucleosides. In another embodiment the second oligomeric compound does not comprise a non hybridizing 3′-overhang region.

The second oligomeric compound comprises a linked conjugate group. A large number of conjugate groups are known to the art skilled that would be amenable to the present invention. Attachment of conjugate groups is also well known in the art and any and all such linkages are envisioned by the present invention. In one embodiment the second oligomeric compound comprises a non hybridizing region, a phosphorothioate group, a linker and a conjugate group. In another embodiment the optional non hybridizing region is omitted. One representative formula for a linked conjugate is shown below for illustration and is not meant to be limiting:

The formula includes a C16 lipophilic conjugate attached via a pyrrolidinyl linker to a phosphorothioate group. The phosphorothioate group can be attached directly to the 3′-end of the second oligomeric compound or can be attached to the 3′-terminal non hybridizing 3′-overhang 2′-modified nucleoside.

The compositions comprising the various motif combinations of the present invention have been shown to have enhanced properties. The properties that can be enhanced include, but are not limited, to modulation of pharmacokinetic properties through modification of protein binding, protein off-rate, absorption and clearance; modulation of nuclease stability as well as chemical stability; modulation of the binding affinity and specificity of the oligomer (affinity and specificity for enzymes as well as for complementary sequences); and increasing efficacy of RNA cleavage.

Independent modification of each nucleoside or groups of nucleosides in each strand allows for maximizing the desired properties of each strand independently for their intended role in a process of gene modulation e.g. RNA interference. Tailoring the chemistry and/or the motif of each strand independently also allows for regionally enhancing each strand. More particularly, the present compositions comprise strands having motifs selected from an alternating motif, a hemimer motif, a blockmer motif, a fully modified motif or a positionally modified motif.

The compositions of the present invention are useful for, for example, modulating gene expression. For example, a targeted cell, group of cells, a tissue or an animal is contacted with a composition of the invention to effect reduction of mRNA that can directly inhibit gene expression. In another embodiment, the reduction of mRNA indirectly upregulates a non-targeted gene through a pathway that relates the targeted gene to a non-targeted gene. Numerous methods and models for the regulation of genes using compositions of the invention are illustrated in the art and in the example section below.

The compositions of the invention modulate gene expression by hybridizing to a nucleic acid target resulting in loss of its normal function. As used herein, the term “target nucleic acid” or “nucleic acid target” is used for convenience to encompass any nucleic acid capable of being targeted including without limitation DNA, RNA (including pre-mRNA and mRNA or portions thereof) transcribed from such DNA, and also cDNA derived from such RNA. In some embodiments, the target nucleic acid is a messenger RNA. In another embodiment, the degradation of the targeted messenger RNA is facilitated by an activated RISC complex that is formed with compositions of the invention. In another embodiment, the degradation of the targeted messenger RNA is facilitated by a nuclease such as RNaseH.

The present invention provides double stranded compositions wherein one of the strands is useful in, for example, influencing the preferential loading of the opposite strand into the RISC (or cleavage) complex. In particular, the present invention provides oligomeric compounds that comprise chemical modifications in at least one of the strands to drive loading of the opposite strand into the RISC (or cleavage) complex. Such modifications can be used to increase potency of duplex constructs that have been modified to enhance stability. Examples of chemical modifications that drive loading of the second strand are expected to include, but are not limited to, MOE (2′-O(CH2)2OCH3), 2′-O-methyl, -ethyl, -propyl, —O—(CH2)2—O—N(CH3)2, —O—CH2C(═O)—N(H)(CH2)2N(CH3)2, —O—(CH2)2—O—(CH2)2—N(CH1)2 and —N-methylacetamide. Such modifications can be distributed throughout the strand, or placed at the 5′ and/or 3′ ends to make a gapmer motif on the sense strand. The compositions are useful for targeting selected nucleic acid molecules and modulating the expression of one or more genes. In some embodiments, the compositions of the present invention hybridize to a portion of a target RNA resulting in loss of normal function of the target RNA.

The present invention provides double stranded compositions wherein one strand comprises an alternating motif and the other strand comprises a hemimer motif, a blockmer motif, a fully modified motif or a positionally modified. Each strand of the compositions of the present invention can be modified to fulfil a particular role in for example the siRNA pathway. Using a different motif in each strand with the same types or different chemical modifications in each strand permits targeting the antisense strand for the RISC complex while inhibiting the incorporation of the sense strand. Within this model each strand can be independently modified such that it is enhanced for its particular role. The antisense strand can be modified at the 5′-end to enhance its role in one region of the RISC while the 3′-end can be modified differentially to enhance its role in a different region of the RISC. Researchers have been looking at the interaction of the guide sequence and the RISC using various models. Different requirements for the 3′-end, the 5′-end and the region corresponding to the cleavage site of the mRNA are being elucidated through these studies. It has now been shown that the 3′-end of the guide sequence complexes with the PAZ domain while the 5′-end complexes with the Piwi domain (see Song et al., Science, 2004, 305, 1434-1437; Song et al., Nature Structural Biology, 2003, 10(12), 1026-1032; Parker et al., Letters to Nature, 2005, 434, 663-666).

As used in the present invention the term “alternating motif” is meant to include a contiguous sequence of nucleosides comprising two different nucleosides that alternate for essentially the entire sequence of the oligomeric compound. The pattern of alternation can be described by the formula: 5′-A(-L-B-L-A)n(-L-B)nn-3′ where A and B are nucleosides differentiated by having at least different sugar groups, each L is an internucleoside linking group, nn is 0 or 1 and n is from about 7 to about 11. This permits alternating oligomeric compounds from about 17 to about 24 nucleosides in length. This length range is not meant to be limiting as longer and shorter oligomeric compounds are also amenable to the present invention. This formula also allows for even and odd lengths for alternating oligomeric compounds wherein the 3′ and 5′-terminal nucleosides are the same (odd) or different (even).

The “A” and “B” nucleosides comprising alternating oligomeric compounds of the present invention are differentiated from each other by having at least different sugar moieties. Each of the A and B nucleosides is selected from β-D-ribonucleosides, 2′-modified nucleosides, 4′-thio modified nucleosides, 4′-thio-2′-modified nucleosides, and bicyclic sugar modified nucleosides. The alternating motif includes the alternation of nucleosides having different sugar groups but is independent from the nucleobase sequence and the internucleoside linkages. The internucleoside linkage can vary at each or selected locations or can be uniform or alternating throughout the oligomeric compound.

Alternating oligomeric compounds of the present invention can the designed to function as the sense or the antisense strand. Alternating 2′-OCH3/2′-F modified oligomeric compounds have been used as the antisense strand and have shown good activity with a variety of sense stands. One antisense oligomeric compound comprising an alternating motif is a 19mer wherein the A\'s are 2′-OCH3 modified nucleosides and the B\'s are 2′-F modified nucleosides (nn is 0 and n is 9). The resulting alternating oligomeric compound will have a register wherein the 3′ and 5′-ends are both 2′-OCH3 modified nucleosides.

Alternating oligomeric compounds have been designed to function as the sense strand also. The chemistry or register is generally different than for the oligomeric compounds designed for the antisense strand. When a alternating 2′-F/2′-OCH3 modified 19mer was paired with the antisense strand in the previous paragraph the preferred orientation was determined to be an offset register wherein both the 3′ and 5′-ends of the sense strand were 2′-F modified nucleosides. In a matched register the sugar modifications match between hybridized nucleosides so all the terminal ends of a 19mer would have the same sugar modification. Another alternating motif that has been tested and works in the sense strand is β-D-ribonucleosides alternating with 2′-MOE modified nucleosides.

As used in the present invention the term “fully modified motif” is meant to include a contiguous sequence of sugar modified nucleosides wherein essentially each nucleoside is modified to have the same sugar modification. The compositions of the invention can comprise a fully modified strand as the sense or the antisense strand with the sense strand preferred as the fully modified strand. Suitable sugar modified nucleosides for fully modified strands of the invention include 2′-F, 4′-thio and 2′-OCH3 with 2′-OCH3 particularly suitable. In one aspect the 3′ and 5′-terminal nucleosides are unmodified.

As used in the present invention the term “hemimer motif” is meant to include a sequence of nucleosides that have uniform sugar moieties (identical sugars, modified or unmodified) and wherein one of the 5′-end or the 3′-end has a sequence of from 2 to 12 nucleosides that are sugar modified nucleosides that are different from the other nucleosides in the hemimer modified oligomeric compound. An example of a typical hemimer is a an oligomeric compound comprising β-D-ribonucleosides that have a sequence of sugar modified nucleosides at one of the termini. One hemimer motif includes a sequence of β-D-ribonucleosides having from 2-12 sugar modified nucleosides located at one of the termini. Another hemimer motif includes a sequence of β-D-ribonucleosides having from 2-6 sugar modified nucleosides located at one of the termini with from 24 being suitable.

As used in the present invention the term “blockmer motif” is meant to include a sequence of nucleosides that have uniform sugars (identical sugars, modified or unmodified) that is internally interrupted by a block of sugar modified nucleosides that are uniformly modified and wherein the modification is different from the other nucleosides. More generally, oligomeric compounds having a blockmer motif comprise a sequence of β-D-ribonucleosides having one internal block of from 2 to 6, or from 2 to 4 sugar modified nucleosides. The internal block region can be at any position within the oligomeric compound as long as it is not at one of the termini which would then make it a hemimer. The base sequence and internucleoside linkages can vary at any position within a blockmer motif.

As used in the present invention the term “positionally modified motif” is meant to include a sequence of β-D-ribonucleosides wherein the sequence is interrupted by two or more regions comprising from 1 to about 4 sugar modified nucleosides. The positionally modified motif includes internal regions of sugar modified nucleoside and can also include one or both termini. Each particular sugar modification within a region of sugar modified nucleosides is variable with uniform modification desired. The sugar modified regions can have the same sugar modification or can vary such that one region may have a different sugar modification than another region. Positionally modified strands comprise at least two sugar modified regions and at least three when both the 3′ and 5′-termini comprise sugar modified regions. Positionally modified oligomeric compounds are distinguished from gapped motifs, hemimer motifs, blockmer motifs and alternating motifs because the pattern of regional substitution defined by any positional motif is not defined by these other motifs. Positionally modified motifs are not determined by the nucleobase sequence or the location or types of internucleoside linkages. The term positionally modified oligomeric compound includes many different specific substitution patterns. A number of these substitution patterns have been prepared and tested in compositions.

Either or both of the antisense and the sense strand of compositions of the present invention can be positionally modified. In one embodiment, the positionally modified strand is designed as the antisense strand. A list of different substitution patterns corresponding to positionally modified oligomeric compounds illustrated in the examples are shown below. This list is meant to be instructive and not limiting.

ISIS No: Length Substitution pattern 5′-3′ Modified positions 345838 19mer 5-1-5-1-2-1-2-2 6, 12, 15 and 18-19 352506 19mer 5-2-2-2-5-3 7-8, 10-11, 17-19 352505 19mer 4-1-2-1-2-1-2-1-2-3 5, 8, 11, 14, 17-19 xxxxxx 19mer 4-1-6-1-4-3 5, 12, 17-19 xxxxxx 19mer 4-2-4-2-5-2 5-6, 11-12, 18-19 345839 19mer 4-2-2-2-6-3 5-6, 9-10, 17-19 xxxxxx 19mer 3-1-4-1-4-1-3-1-1 4, 9, 14, 18 353539 19mer 3-5-1-2-1-4-3* 1-3, 9, 12 355715 19mer 3-1-4-1-8-1-1 4, 9, 18 xxxxxx 19mer 3-1-5-1-7-1-1 4, 10, 18 384760 19mer 2-7-2-5-3* 1-2, 10-11 and 17-19 371315 19mer 3-6-2-5-3 1-3, 10-11, 17-19 353538 19mer 2-1-5-1-2-1-4-3 3, 9, 12, 17-19 xxxxxx 19mer 2-1-4-1-4-1-4-1-1 3, 8, 13, 18 336674 20mer 15-1-1-3 16, 18-20 355712 20mer 4-1-2-1-2-1-2-1-2-3* 5, 8, 11, 14 347348 20mer 3-2-1-2-1-2-1-2-1-2-3 1-3, 6, 9, 12, 15, 18-20 348467 20mer 3-2-1-2-1-2-1-2-1-5 1-3, 6, 9, 12, 15 357278 20mer 3-1-4-1-4-1-3-1-1 4, 9, 14, 18 xxxxxx 20mer 3-1-1-10-1-1-3 1-3, 5, 16, 18-20 xxxxxx 20mer 3-1-6-1-7-1-1 4, 11, 19 357276 20mer 3-1-3-1-7-1-4 4, 8, 16 xxxxxx 20mer 3-1-5-2-5-1-3 4, 11, 17 357275 20mer 3-1-5-1-8-1-1 4, 10, 19

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